In vivo anti-inflammatory, analgesic activities and phytochemical profile of Hu Lisan ethanol extract

Huli San (HLS) is a classic compound formulation long utilized by ethnic minorities such as the Yi and Miao peoples in Southwest China. Its composition blends traditional medicinal herbs including Aconitum vilmoriniannum Kom (AK), Panax notoginseng (Burk.) F. H. Che (PN), Cynanchum wallichii Wight (CW), and Campanumoea javanica BI (CJ), creating a formula deeply rooted in the rich tradition of ethnic pharmacology. This compound is renowned for its ability to dispel wind and dampness, invigorate blood, and alleviate pain. It is commonly used to treat rheumatic pain, numbness of muscles and bones, and injuries from falls and bruises, reflecting the indigenous understanding of the interplay between "wind-damp-stasis" as the core pathological mechanism. Traditionally, Huli San is prepared through processes such as grinding, resulting in either a powder or tincture for oral administration. Its clinical efficacy is centered around the relief of wind-damp conditions, promoting blood circulation, and alleviating pain, particularly in the treatment of rheumatic disorders, musculoskeletal numbness, and traumatic injuries.

The study aimed to comprehensively analyze the chemical components, acute toxicity, as well as anti-inflammatory and analgesic effects of the HLS ethanol extract (HLS-EE) and compare it with the commercial drug Hulisan capsules (HLS-C).

The chemical composition of HLS-EE was characterized using UPLC-MS/MS, and its median lethal dose (LD₅₀) was determined to establish a safe dosage range. Toxicological assessment included observation of pathological alterations in major organs. The anti-inflammatory efficacy of HLS-EE and HLS-C was evaluated utilizing three classical models: xylene-induced ear edema, carrageenan induced paw edema, and acetic acid-induced vascular permeability in mice. Their analgesic activity was assessed via the acetic acid-induced writhing test, hot plate test, and formalin-induced biphasic pain test.

The UPLC-MS/MS analysis showed that there were 156 kinds of chemicals in HLS-EE. The LD₅₀ value is 2.962 g/kg. Histopathological examination showed no major abnormalities in the heart, liver, spleen, lungs, and kidneys at 15 times the clinical dose (CLD); at 90 times and 120 times the CLD, there were inflammatory cell infiltrations in multiple organs. HLS-EE showed great activity on anti-inflammatory testing. It significantly inhibited xylenc-caused ear swelling, carragenan-caused paw swelling and aceticacid-caused vascular leakage tests. Middle and high-dose HLS-EE groups exhibited stronger inhibition than HLS-C. Mechanistically, HLS-EE reduced tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) in serum from ear edema model.In the paw edema model, it decreased IL-1β, IL-6, TNF-α, prostaglandin E₂ (PGE₂), nitric oxide (NO), and malondialdehyde (MDA), while increasing superoxide dismutase (SOD) activity. Additionally, HLS-EE downregulated protein and mRNA expression of cyclooxygenase-2 (COX-2), IL-6, TNF-α, and extracellular signal-regulated kinase _1/2 (Erk_1/2) in paw tissues. In analgesic tests, HLS-EE markedly reduced writhing responses, prolonged thermal pain latency, and suppressed formalin-induced biphasic pain, with middle and high doses outperforming HLS-C. Serum analysis showed decreased 5-hydroxytryptamine (5-HT), IL-1β, and PGE₂ in writhing-test mice and reduced IL-1β and IL-6 in formalin-test rats. Spinal cord analysis further indicated downregulated mRNA expression of cellular oncogene fos (c-Fos), 5-hydroxytryptamine 2A receptor (5-HT_2A), kappa opioid receptor (KOR), and prodynorphin (PDYN), suggesting central analgesic mechanisms.

A total of 156 chemical components were identified in HLS-EE. The LD₅₀ of HLS-EE is 2.962 g/kg. HLS-EE exhibits good anti-inflammatory and analgesic effects, which are superior to those of the commercially available HLS-C. This study provides a basis for further research and formulation development of HLS-EE and contributes to the promotion of the development and utilization of ethnic medicine.